Class Notes (838,950)
Canada (511,158)
UOIT (1,700)
Biology (294)
BIOL 2050U (48)


5 Pages
Unlock Document

BIOL 2050U
Peter Cheung

GENETIC BASIS OF ANTIBODY DIVERSITY -Constant portion alleles are encoded on a single locus. -Variable regions have multiple loci!!! Structural basis of antigen binding 3D Structure of Ig Domains - Antibody sequence variability is clustered variability = # of different observed aa’s/ frequency of most common range = 1(least) – 400(most) observe clustering in 3 HYPERVARIABLE regions interspersed by FRAMEWORK regions true for light and heavy chains **hypervariable regions involved in antigen binding** also called CDR’s (complementarity determining regions) 3D structure of antibody domains: all domains have IMMUNOGLOBULIN fold all fold ind. into a globular domain and there is interaction betweeC H andCL (V Hnteracts w/ V L Three hypervariable regions brought together as three loops that extend from ends of VH and VL Hypervariable regions cap the distal end 3D structures of antibody/ligand binding: 1. larger proteins  more interaction (more CDR’s involved) 2. framework residues contact somewhat 3. epitope (surface of antigen that contacts Ab) composed of noncontiguous AA residues 3. large interacting surfaces w/ high STERIC complementarity 4. hydrophobics/h-bonds are V. important 5. LITTLE conf. change (saves energy) EPITOPE = minimal amt of antigen needed to bind antibody GENERATION OF VARIABLE REGION DIVERSITY BY V(D)J RECOMBINATION -kappa genes are on single genetic locus but associate w/different Vregions -V regions are encoded by multiple gene segments (somatic recombination) HC and LC are coded PIECEWISE- brought together by site specific recomb. during B-cell differ. Light Chain Genes: One Recombination Igk V and J separated in germline, joined during lymph. Diff. (somatic recombination DNA) Intron btw Jk and Ck spliced after transcription (RNA splicing) Igλ have multiple Jλ-C λ Heavy Chain Genes: (more important for variability): *3 Gene Segments: VDJ (2 somatic recombinations) *J Hluster separated from Cμ by an intron *The exons encoding C reHions are arranged in tandem Detection of Ab gene Rearrangements: Southern blotting 1. DNA from Ab producing cell cut with restriction enzyme, fractionated by gel el, transfer to membrane 2. Hybridize w/DNA probe specific for an Ab gene segment 3. Rearrangement detected as appearance of new fragment and decrease of unrearranged DNA fragment -use oligonucleotide primers specific for the pairs of gene segments under study -amplified product only rearranged if segments with primers are joined Mechanism of Antibody Gene Assembly -RSS (recombination signal sequences) mediates assemble of antibody genes -flank unrearranged Ab gene segments -highly conserved 7 and 9 bp separated by 12 or 23 bp (less conserved) -gene segments with RSS with 12 spacer only recombines with 23 spacer 12-23 RULE!! -V Hegments (23) only recombine with D segments (12)!!! V(D) J recombination -RSS are recognition sites for RECOMBINASE proteins (RAG1/2) Only in Developing Lymp 1. check to be sure spacer sequences are different 2. initiate ds DNA cleavage btw RSS and gene segment -blunt signal end -coding end w/closed hairpin structure -Then a protein joins the coding and signal ends (scid mutantsno B or T cells) RSS are spliced out; coding sequences are joined If V and J in the same directionloop excised Opposite directionlinear first RAG mechanism: transesterification 1) nick one one strand where RSS and coding sequence meet frees a hydroxyl 2) hydroxyl at 3’ attacks the OPPOSITE strand phosphate leaves a hairpin (coding) and blunt end (RSS end) **very similar to mechanism of bacterial transposition and retroviral integrases** Difference: Target is a P group on the opposite strand of the same DNA molecule (not different DNA mol) But invitro RAG performs transposition 3) If trimming at site of cleavage by ARTEMIS (activated by DNA-PK )(recruited by Ku) TdT (terminal deoxynucleoitidyl) adds extra nucleotides AT RANDOM into junction before ligation “junctional diversity” N regions – created in H usually, L sometimes C C - not encoded in germline, highly variable in length 3) if no trimming occurs  -before coding ends can be joined, they are cut open by a nuclease DNA Pol. inserts complementary nucleotides PALINDROMIC with the ends of coding sequences (P Element Insertion) FINAL PRODUCT: VPN **N REGIONS AND P ELEMENTS ARE PART OF CDR3** 9 TOTAL POSSIBLE: 10 different antibodies (includes combinatorial and junctional diversity) SCID (severe combined immunodeficiency) – due to RAG defects no ds breaks generated CLASS SWITCHING
More Less

Related notes for BIOL 2050U

Log In


Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.